Studies in a Tumor Spectrum III. The Effect of …...Studies in a Tumor Spectrum III. The Effect of...

Studies in a Tumor Spectrum

III. The Effect of Phosphor amides on the Growthof a Variety of Mouse and Rat Tumors*

KANEMATSUSUGIURAANDC. CHESTERSTOCKWITHTHEASSISTANCEOFMITONOM. SUGIURA

(Laboratories of the Sloan-Kettering Division of Cornell University Medical Collegein the Division ofExperimental Chemotherapy, Sloan-Kettering Institute for Cancer Research, New York, N. Y.)

Previous investigations with a spectrum of tumors demonstrated that nitrogen mustards (17)and triethylene melamine (19) had a definite inhibitory or destructive action on certain types oftumors. In view of the fact that the biological activity of these compounds is believed to be due tothe formation of ethylenimmonium ions in thecase of the former (7) and the presence of ethyleni-mine groups in the case of the latter, the study wasextended with compounds closely related to thesesubstances on a wide spectrum of tumors.

Results in experimental animal tumors reportedfrom other laboratories (1-3, 6, 9-11, Â£0,21) andin human cancer (4, 8, 20) have indicated that triethylene phosphoramide and triethylene thiophos-phoramide merited intensive study. Our observations on the effect of these compounds on a varietyof transplantable mouse and rat tumors have beenbriefly reported (14, 18) and are now presented inmore detail. The structural formulae of these compounds have in common the cyclic structure,ethylenimine :

/\HSC2 C2HÂ»

l\HtC-CHÂ£ f\HgC-CHj

CM,

OEPA TE PA TSPA

DEPA= N,N-Diethyl-N',N"-diethylene phosphoramide

TEPA = N,N',N"-Triethylene phosphoramide

TSPA = N,N',N"-Triethylene thiophosphoramide

MATERIALS AND METHODSThe tumors used in the present study are as fol

lows: Sarcoma 180 (solid and ascitic forms), Bash-

* This study was supported by an institutional grant to theSloan-Kettering Institute from the American Cancer Societyand by a grant from the Damon Runyon Memorial Fund forCancer Research.

Received for publication August S, 1954.

ford carcinoma 63, Ehrlich carcinoma (solid andascitic forms), Krebs 2 ascites carcinoma andHarding-Passey melanoma in Rockland Swiss albino mice; Sarcoma T 241, Adenocarcinoma E0771, Lewis bladder carcinoma and Lewis lungcarcinoma in C57BL mice; Sarcoma MA 387,Miyono adenocarcinoma, Carcinoma 1025, Wagner osteogenic sarcoma, Ridgway osteogenic sarcoma, Patterson lymphosarcoma and Mecca lympho-sarcoma in AKR mice; Grand epidennoid carcinoma in CFW mice; Gardner lymphosarcoma inC3H mice; Andervont hepatoma in C mice; Flex-ner-Jobling carcinoma, Walker carcinosarcoma256, and Sarcoma R 39 in Sherman rats; Jensensarcoma in Sprague-Dawley rats; and Murphy-Sturm lymphosarcoma in Wistar rats.

The history, biological properties, and cytologi-cal description of most of these tumors were presented previously (17, 19). Similar descriptions ofother tumors used in this paper are given below.1

Grand mouse epidermoid carcinoma.â€”Thistumor was discovered September 7, 1951, by C. G. Grand at New York University. It originated spontaneously in the skin of a CFWmouse. It was first cultivated in tissue culture and retrans-planted into CFW mice. The histological picture of the originaltumor was that of a squamous-cell carcinoma (Fig. 1). Thetumor shows occasional squamous pearl formation. The tumorgrows rapidly, and 14 days after transplantation into CFWmice it reaches a size of approximately 15 X 12 X 8 mm. Thistumor becomes extensively necrotic and cystic in the centerafter 14 days. The tumor transplants take in 100 per cent ofcases, and about 30 per cent of them regress. The 76th transplantation generation was reached on June 10, 1954.

Leids mouse bladder carcinoma?â€”This transplantable

1The authors wish to acknowledge their indebtedness to thefollowing individuals for supplying the original tumors andtheir histories: Dr. Margaret R. Lewis of the Wistar Institutefor the mouse bladder carcinoma and mouse lung carcinoma;Mr. C. G. Grand, Cancer Institute at Miami, for the mouseepidermoid carcinoma.

1Subcutaneous or intraperitoneal injection of fresh blood ofmice bearing the Lewis bladder carcinoma or Lewis lungcarcinoma into mice produced identical tumors. Dr. MargaretR. Lewis, personal communication, 1954.

88

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SuGiURAANDSTOCKâ€”Phosphoramidesin a Tumor Spectrum 39

tumor was discovered in a male C57BL mouse in December,1950. The tumor is a very malignant type of epidermoid carcinoma (Fig. 2). It grows rapidly and becomes very hemor-rhagic; 14 days after transplantation into C57BL mice, itreaches a size of approximately 19 X 13 X 10 mm. The transplants take in 100 per cent of cases, and about 2 per cent ofthem regress. In our laboratory the 16th transplantation generation was reached on July 5, 1954.

Lewis moine lung carcinoma*â€”This tumor originatedspontaneously as a carcinoma of the lung of a C57BL mousein 1951. It is a rapidly growing tumor, reaching a size of approximately 18 X 12 X 9 mm. 14 days after transplantation.The transplants take in 100 per cent of cases, and about 4 percent of them regress. The tumor is a very malignant type ofepidermoid carcinoma (anaplastic carcinoma) and becomesextremely hemorrhagic (Fig. 3). In our laboratory the 16th

^r transplantation generation was reached on July 6, 1954.The methods employed in the chemotherapy studies of the

tumors have been described before (16). In general, subcutaneous implantations of tumor material (small pieces of tumor,each measuring about 1.5 mm. in any dimension and weighingapproximately 6 mg.) into healthy young animals (18-22-gm.mice; 100-125-gm. rats) were carried out by the usual trocarmethod (a single implant into the right axillary region).

In every set of experiments, tumor-bearing animals weredivided into two groups, one to be treated with compoundsand the other to be used as controls. The progress of the tumorsin the animals was recorded graphically by measuring them intwo diameters with calipers at the end of 1, 2, 3, and 4 weeksafter tumor transplantation. The final measurements of thetumors were made at the end of the 6th week. In the case ofascites tumors (13), intraperitoneal injection of 0.1 cc. of thefluid containing about 1 million cancer cells into mice was madein the inguinal region with a i-cc. syringe having a 23-gaugeneedle.

The first intraperitoneal injection of the compounds wasgiven either 1 or 7 days after tumor transplantation. Freshsaline solutions of DEPA, TEPA, and TSPA were prepareddaily, and 0.5 cc. was injected once daily for 7 days. The animals were given food (Purina Laboratory Chow) and waterad libitum.

The degree of inhibition of tumor growth was graded according to the following scheme:

â€” No effect; tumor growth to three-quarters or more ofthe diameter of the controls.

Â± Slight inhibition; tumor growth from one-half tothree-quarters of the diameter of the controls.

+ Moderate inhibition; tumor growth from one-fourthto one-half of the diameter of the controls.

-1â€”(-Marked inhibition; failure to grow or growth to approximately one-fourth of the average diameter of thecontrols.

Hâ€”h+ Complete destruction of tumors (Complete regression of a higher percentage of tumors in the treatedanimals than in the controls which show no more than10 per cent spontaneous regressions with the exception of the Grand epidermoid carcinoma, which has30 per cent).

The degree of inhibition of ascites tumor growth was gradedaccording to the following scheme:No effect (â€”)indicates marked abdominaljdistention, or 4

times the normal gain in body weight or more.Slight inhibition ( Â±) indicates moderate abdominal distention.Moderate inhibition (+) indicates slight abdominal distention,

or 2 times the normal gain in body weight.Marked inhibition (-)â€”(-)indicates no abdominal distention,

or normal gains in body weight of animalâ€”about 0.5gm/day.Â»

In all the tables one column gives average weight change ingrams of treated animals over control animals after 1 week oftreatment. All the results with Sarcoma 180 are evaluated onlyon the basis of Ist-week results.

RESULTSGeneral findings.â€”Tables 1-6 summarize the ef

fects of DEPA, TEPA, and TSPA on the growthof eighteen tumors of the mouse and five tumors ofthe rat. With the exception of the Andervont hepa-toma, controls of these experiments showed 100per cent takes and, except for the Grand epidermoid carcinoma, there was a low rate of regressions. The average percentages of tumor regressions in control animals were as follows: Sarcoma180, 2; Sarcoma T 241, 0; Sarcoma MA 387, 6;adenocarcinoma E 0771, 2; Bashford carcinoma63, 6; Miyono adenocarcinoma, 6; Ehrlich carcinoma, 0; Carcinoma 1025, 4; Grand epidermoid carcinoma, 30; Wagner osteogenic sarcoma, 7; Ridg-way osteogenic sarcoma, 7; Patterson lymphosar-coma, 3; Mecca lymphosarcoma, 4; Gardner lym-phosarcoma, 4: Harding-Passey melanoma, 6; Andervont hepatoma, 0; Lewis bladder carcinoma, 0;Lewis lung carcinoma, 4; Flexner-Jobling carcinoma, 10; Walker carcinosarcoma 256, 3; SarcomaR 39, 7; Jensen sarcoma, 6; and Murphy-Sturmlymphosarcoma, 5 per cent.

The compounds have been studied at a numberof dose levels in an attempt to obtain data at maximum tolerated doses or at comparable degrees oftoxicity. It is apparent from the data that bothTEPA and TSPA at their maximum tolerateddoses have shown a similar degree of definite inhibitory or destructive actions upon certain of thetransplantable tumors. The mouse tumor most affected by TSPA was Carcinoma 1025. Completedestruction of 1-day-old tumors resulted from dailydoses of 4 mg/kg or 2 mg/kg in 3 weeks (Chart 1).DEPA was considerably less effective, though itdid cause a marked retardation in the growth ofCarcinoma 1025 and the Ridgway osteogenicsarcoma. The decreased effectiveness of DEPAwas also apparent in tests with the ascites formsof the tumors.

It is extremely interesting that repeated injections of TSPA had a marked inhibitory effect onHarding-Passey melanoma. We have tested over500 other compounds with this tumor, and theycaused only slight or no inhibitory effect.

ResidÃs with spontaneous mammary cancers.â€”Since TEPA and TSPA had a distinct inhibitoryeffect on the growth of four transplantable mam-

3In some animals the daily weight increase may be muchless due to toxic action of compounds. Survival of more than 50per cent of treated an mais beyond 3 weeks was considered asa marked effect. Control animals die in 2-3 weeks.



TABLEl.â€”EFFECTOFDIETHYLENEPHOSPHORAMIDEONVARIOUSI-ÃœAY-OLDMOUSETUMORS(dose: 4 mg/kg/day)*

TUMOR

Sarcoma 180 (solid)

No. DEATHS

1st week id week1/20 6/20

Av. WT. CHANGE

TREATED/CONTROLS RESULTS OF TREATMENT

Sarcoma 180 (ascitic) 0/20 10/20

Sarcoma T 241 0/15 1/15

Sarcoma MA 387 0/20 1/20

Adenocarcinoma E 0771 0/20 0/20

Bashford carcinoma 63 0/25 2/25

Miyono adenocarcinoma 0/30 1/30

Ehrlich carcinoma (solid) 0/15 0/15

Ehrlich carcinoma (ascitic) 0/30 10/30

Krebs 2 carcinoma (ascitic) 0/30 17/30

Carcinoma 1025 0/20 0/20

Grand epidermoid carcinoma 0/15 0/15

Wagner osteogenic sarcoma 2/10 6/10

Ridgway osteogenic sarcoma 0/40 1/40

Patterson lymphosarcoma 0/25 6/25

Mecca lymphosarcoma 0/20 0/20

Gardner lymphosarcoma 0/20 1/20

Harding-Passey melanoma 0/25 0/25

Andervont hepatoma 0/10 0/10

Lewis bladder carcinoma 0/10 0/10

Lewis lung carcinoma 0/10 2/10

(gm.)0

+2Â±5+8-1+ 1-1+2-0.5

+1+0.5+2-1

1st week id week

+ 1.5

+1+3

0+4~5

+7

+8.5-1.5

+ 1.5

0+1.5-2

+ 1.5-0.5

+1-2

+ 1-1

+2-0.5

+TS+ 1.5

+4-4

+Ã•-1

+1-15

REMARKS

Normal growth thereafter


90 per cent inhibition at19th week

+ 1.5* Five or ten animals in each test group. The results presented are the averages of combined groups.

TABLE2.â€”EFFECTOFDIETHYLENEPHOSPHORAMIDEONVARIOUSI-DAY-OLDRATTUMORS(dose: 1 mg/kg/day)*

Av. WT. CHANGENo. DEATHS TREATED/CONTROLS RESULTS OF TREATMENT

1st week id week (gm.) 1st week id week

0/25 1/25 +7 + ++ +TUMOR

Flexner-Jobling carcinoma

Walker carcinosarcoma 256

Sarcoma R 39

Jensen sarcoma

0/35 2/35

2/25 6/25

0/20 3/20

Murphy-Sturm lymphosarcoma 0/20 4/20

+25+9

+26+17+23+14+25+15

REMARKS47 per cent inhibition at 2d week;

75 per cent at 3d week

15 per cent inhibition at 2d week;29 per cent at 3d week


100 per cent inhibition at 2d and3d week

+27* Five or ten animals in each test group. The results presented are the averages of combined groups.



SuGiURAANDSTOCKâ€”Phosphoramidesin a Tumor Spectrum 41

mary carcinomas, TSPA was therefore tested in a breast tumors, but there was no significant in-total of 80 Rockland Farms Swiss albino mice with crease in tumor regression over the 7 per cent ob-recently developed spontaneous mammary adeno- served in the controls. This suggests that the regres-carcinomas. In various groups of these mice injec- sions observed with well developed tumors aftertions of TSPA (2 or 4 mg/kg/day for 10-18 days) treatment with TEPA or TSPA may result fromstopped the growth of the majority of spontaneous extensive initial damage from the drug followedbyTABLE

3EFFECTOFTRIETHYLENEPHOSPHORAMIDEONVARIOUSI-DAY-OLDMOUSETUMORS

(dose:6mg/kg/day)*AV.

WT. CHANGE

No. DEATHS TBEi TED/CONTROLS RESULTS OFTREATMENTTUMOR

1stweek 2dweek (gm.) lit week 4d week REMARKSSarcoma 180 (solid) 0/25 5/25 -1 ++ + Normal growth thereafter

+2Sarcoma 180 (ascites) 0/20 5/20 +1.5 +Â±Sarcoma

T 241 0/20 5/20

Sarcoma MA 387 0/20 6/20

Adenocarcinoma E 0771 0/20 2/20

Bashford carcinoma 63 0/15 1/15

Miyono adenocarcinoma 0/20 4/20

Ehrlich carcinoma (solid) 0/20 4/20

Ehrlich carcinoma (ascites) 0/40 9/40

Krebs 2 carcinoma (ascites) 0/20 2/20

Krebs 2 carcinoma (ascites) 0/10 2/10

Carcinoma 1025 0/40 7/40

Grand epidermoid carcinoma 2/20 2/20

Wagner osteogenic sarcoma 2/30 9/30

Ridgway osteogenic sarcoma 0/30 5/30

Patterson lymphosarcoma 0/25 6/25

Mecca lymphosarcoma 0/30 9/30



Harding-Passey melanoma 0/20 1/20

Andervont hepatoma 0/20 0/20

Lewis bladder carcinoma 1/20 4/20

Lewis lung carcinoma 0/20 2/20+9

-1.5++

1-2Â±+1.5

-2.5Â±+1

-1.5-+2.5

-3.5 +++

1-1.5++4

-1 +++6.5

-1.5+++8

_0_t ++-3

+++1.5

-1.5++1.5-3

+

+1-3.5 +++1.5

-2.5++1.5

-2.5 +++1.5

-3 +++1-It+++2

+1-3

?+0.5

-1.5Â±+1.5

-2.5 Â±Â±

+ Normal growth thereafter

Â±

+ + Normal growth thereafter

+ Normal growththereafter++

100 per cent inhibition in groupat 2dweekH

â€”|â€”h 42 per cent inhibition at 2d week;100 per cent at 3dweek+


+ Normal growththereafter+

Hâ€”h 70 per cent inhibition at 2d week;85 per cent at 3dweek+


+ Normal growth thereafter

Hâ€”h 25 per cent inhibition at 3d week

+ + Normal growththereafterÂ±?

65 per cent inhibition at 19thweek+


+ Normal growth thereafter+1

* Five or ten animals in each test group. The results presented are the averages of combined groups.t 4 mg/kg/day.



Cancer Research

immunological reactions of the host. The latterpresumably would not come into play after damage to spontaneous tumors, even though that damage be extensive enough to stop growth of the established tumor during treatment.

Toxicity.â€”Extensive studies on the pharmacol

ogy of DEPA and TEPA in normal animals havebeen reported (20). The following summarizes thetoxicity data of TEPA at different dosages: at 10,6, and 4 mg/kg/day for 7 days, 25, 1.5, and 0 percent, respectively, of tumor-bearing mice died during treatment. At 6 mg/kg/day, the animals lostbody weight; the weight loss was extensive athigher levels. At 4 mg/kg/day for 7 days, therewere no deaths, and the mice maintained body

below normal. At 0.5 mg/kg/day or less all ratsgrew normally.

Results with rat tumors.â€”The greater toxicity of

DEPA, TEPA, and TSPA for rats was paralleledby their greater capacity to produce damage insome of the rat tumors, as has been previously reported (16,17,19) for other compounds. The phos-phoramides were most effective in Sarcoma R 39and Jensen sarcoma and decreasingly effective inthe Flexner-Jobling carcinoma, Walker carcino-sarcoma 256, and the Murphy-Sturm lympho-sarcoma. At a dose of 1 mg/kg/day of TEPA for7 days, for example, the growth of 1-day-old implants of tumors in that order was prevented asfollows : 100, 90, 64, 83, and 0 per cent. At a dose of

TUMORFlexner-Jobling carcinoma

TABLE 4EFFECTOFTRIETHYLENEPHOSPHORAMIDEONVARIOUSI-DAY-OLDRATTUMORS

(dose: 1 mg/kg/day)*

Av. WT. CHANGENO. DEATHS TRKATED/CONTBOLS RESULTS OF TREATMENT

1st week id week (gm.) 1st week

+5 ++26

id week


Sarcoma R 39

Jensen sarcoma

Murphy-Sturm lymphosar-coma

1/30

0/30

1/20

0/30

1/30

5/30

3/30

1/20

0/30

4/30

+ 1

+23+9+ 19+ 15+27

+ +

+ +

+ + +

+ + +

REMARKS





+32

* Ten animals in each test group. The results presented are the average of combined groups.

weight or gained weight. Rats tolerated the compound less well than mice, 16 per cent dying from5 mg/kg/day for 7 days. At 1 mg/kg/day onlyabout 2 per cent of the rats died. Although therewas no gain in body weight for 1 week, there wasnormal growth thereafter.

The following summarizes the toxicity data ofTSPA at different dosages: at 8, 4, 2, and 1 mg/kg/day for 7 days, 50, 1, 0, and 0 per cent, respectively, of tumor-bearing mice died during treatment. At 4 mg/kg/day the animals lost weight for2 weeks, and those without tumors grew normallythereafter. At 2 mg/kg/day the animals maintained body weight for 7 days; there was normalgrowth thereafter. At 1 mg/kg/day for 7 daysthere was normal gain in body weight. Rats tolerated TSPA less than mice, 50 per cent dying from4 mg/kg/day for 7 days. At 2 mg/kg/day onlyabout 1 per cent of rats died and, while there wasno gain in body weight for 1 week, there was normal growth thereafter. Histological examination ofthe spleen, bone marrow, and organs of animalsshowed no abnormality. At daily doses of 1 mg/kg/day there was gain in body weight, but it was

1 mg/kg/day of DEPA, the following figures wereobtained: 100, 100, 75, 29, and 0 per cent. At adose of 2 mg/kg/day of TSPA, the following figures were obtained: 100 for Jensen sarcoma, 100for Flexner-Jobling carcinoma, 67 for Walker carcinosarcoma 256, and 0 per cent for Murphy-Sturm lymphosarcoma. No figures were availablefor Sarcoma R 39, because the tumor was contaminated and lost. Chart 2 illustrates the effectof TSPA on the Jensen sarcoma. Following sevendaily doses of 2 mg/kg of TSPA, all original 1-day-old tumors were destroyed by the end of 2 weeks.TSPA at a dose of 0.5 mg/kg/day or one-fourth ofthe maximum tolerated dose had complete destructive effects, but at a dose of 0.25 mg/kg orone-eight of a maximum tolerated dose, the compound had only a moderate inhibitory effect onthis tumor. It is interesting to note that sevenadditional injections at a dose of 0.25 mg/kg resulted in marked inhibition of growth, but therewas no significant amount of tumor destruction.

Results with well established mouse and rat tumors.â€”The success of these phosphoramides inproducing damage in 1-day-old tumors of the



TABLE5EFFECTOFTRIETHYLENETHIOPHOSPHORAMIDEONVARIOUSi-DAY-OLDMOUSETUMORS

(dose: 4 mg/kg/day)*

AT. WT. <HANGE

TUMOSSarcoma

180 (solid)

Sarcoma 180 (ascites)

Sarcoma T 241

Sarcoma MA 387

Adenocarcinoma E 0771

Bashford carcinoma 63

Bashford carcinoma 63

Miyono adenocarcinoma

Ehrlich carcinoma (solid)

Ehrlich carcinoma (ascitic)

Krebs 2 carcinoma (ascitic)

Carcinoma 1025

Carcinoma 1025

Grand epidermoid carcinoma

Wagner osteogenic sarcoma

Ridgway osteogenic sarcoma

Ridgway osteogenic sarcoma

Patterson lymphosarcoma

Mecca lymphosarcoma

Gardner lymphosarcoma


Harding-Passey melanoma

Harding-Passey melanoma

Andervont hepatoma

Lewis bladder carcinoma

Lewis lung carcinomaNo.

DEATHS TREATED/CONTROLS RESULTSOP1st

week id week (gm.) 1st week0/20 0/20 -1.5+0/20

20/20

1/20 5/20

0/20 3/20

8/40 6/40

0/80 1/30

0/20 0/20

0/80 5/30

0/20 3/20

0/30 9/30

0/20 10/20

0/40 7/40

0/20 2/20

0/20 0/20

1/40 3/40

0/40 4/40

0/10 0/10

0/20 1/20

1/40 15/40

0/40 2/40

0/40 0/40

2/50 3/50

0/20 0/20

0/20 6/20

0/30 3/30

1/20 2/20+3

+1.5++3.5-2

++1.5

-1.5Â±+2.5

-2 +

+1+0.5++5+2.5

-1.5++1.5

-2.5Â±+2.5

+2+++7.5

+1 ++8-2.5 +++1.5

-It+++2.5

-1 Â±+3-2Â±+1.5

-2 Â±

+1otÂ±+1.5

-2.5++1.5

-2 +

+1-2.5+++1

-It +

+1+0.5-+4.5+0.5f+5

-8 ?

+1-2 Â±

+1-2.5 +TREATMENTid

weekÂ±Â±

Â±+Â±

Â±+1

* Ten animals in each test group. The results presented are the averages of combined groups,

t * mg /kg/day.

REMARES











++ at 3d week



Hâ€”1-at 3d week; retarded growththereafter

Hâ€”hat 3d week; retarded growththereafter

90 per cent inhibition at 8th week





44 Cancer Research

mouse and of the rat led to the more rigorous teston well established tumors. The results obtainedfrom these experiments are summarized in Table 7and presented in detail in Tables 8-10. Treatmentof 7-day-old carcinoma (C1025), osteogenic sarcomas (Ridgway), and lymphosarcomas (Gardner)with 6 mg/kg/day of TEPA or 4 mg/kg/day ofTSPA for 7 consecutive days caused growth tostop for a few days; then the tumors regressedcompletely (about 60-90 per cent of cases) in 2-3weeks. There was no reappearance of tumors dur-

perimental periods of 2-3 months, and on autopsyno material was found at the former site of the tumor. Such cases are recorded as "cures."

In contrast to the findings with untreated tumors (Fig. 4), the histological examination of anumber of the tumors (Jensen sarcomas) removedfrom the host after seven injections of 2 mg/kg ofTSPA showed almost complete necrosis of the tumor cells as seen in the lower right half of Figure 5. The surrounding tissue (upper left half) ismade up of granulation tissue, with a few lympho-

TABLE 6

EFFECTOFTRIETHYLENETHIOPHOSPHORAMIDEONVARIOUSI-ÃœAY-OLDRATTUMORS(dose: 2 mg/kg/day)*

No. DEATHS

TDMOB 1st week id weekFlexner-Jobling carcinoma 1/30 3/30

Flexner-Jobling carcinoma 0/10 1/10

Walker carcinosarcoma 256 1/30 I/SO

Walker carcinosarcoma 256 0/10 0/10

Jensen sarcoma 0/40 0/40







Av. WT. CHANGE

TREATED/CONTROLS RESULTS OF TREATMENT

(gm.)-8

+20+5t+ 15+5+25+lpt+26+0.5+23

1st week Â«dweek

+27+22ÃŽ+26+ 18Â§+25+26#+24

REUARKS


100 per cent inhibition at 2d and3d weeks

50 per cent inhibition at 3d week;67 per cent at 4th week

50 per cent inhibition at 3d week




25 per cent inhibition at 3d week

+18+9f+20

* Ten animals in each test group. The results presented are the averages of combined groups.

11 mg/kg/day.ÃŽ0.5 mg/kg/day.Â§O.Ã•Amg/kg/day.f 0.1

ing experimental periods of 2-3 months, indicatingthe possibility of permanent regression.

At the lower doses of 2 and 1 mg/kg/day, theTSPA was less effective. Thus, seven treatmentswith this compound resulted in only a slight tomoderate inhibition of tumor growth but with nocomplete destruction of 7-day-old tumors.

Treatment of 7-day-old carcinomas (Flexner-Jobling) and sarcomas (R 39 and Jensen) with 1.0mg/kg of TEPA, 1.0-2.5 mg/kg of DEPA, and2.0 mg/kg of TSPA caused growth to stop for afew days and then complete regressions of the tumors (about 70-90 per cent of cases) in 2-3 weeks.There was no reappearance of tumors during ex-

cytes, macrophages, and plasma cells. Tumorswith this histological appearance when transplanted into normal rats do not grow.

The Walker carcinosarcoma 256 did not respond as well to the antitumor activity of TEPAand DEPA as did the Flexner-Jobling carcinoma,Sarcoma R 39, and Jensen sarcoma in the rat. Ata dose of 1.0 mg/kg/day of TEPA and 1.0-2.5mg/kg/day of DEPA for 7 days, the growth of7-day-old tumors was slightly or moderately inhibited but there was no complete tumor regression. TSPA at a level of 2 mg/kg/day had a definite destructive effect on well established 7-day-old Walker carcinosarcoma 256. As indicated in



SuGiURAANDSTOCKâ€”Phospkoramidesin a Tumor Spectrum 45

Tables 8-10, none of the three phosphoramideshad an effect on 7-day-old Murphy-Sturmlymphosarcoma. All tumors grew normally.

The study was continued with lower doses ofTSPA on Flexner-Jobling carcinoma and Jensensarcoma in rats. The results of the experimentsshowed that daily doses of 1 mg/kg of TSPA re-

During the course of the study we determinedthe effect of TSPA on the growth of two differenttumors growing simultaneously in the same rat. Inthis case we selected first Jensen sarcoma andMurphy-Sturm lymphosarcoma. The former issensitive, while the latter is resistant to thecompound.

EFFECT OF TSPA ON CARCINOMA 1025 IN MICE

4 mg/k

14 21

3 â€¢

4 â€¢

5

6 â€¢

7 .

8

9 .

10 .

cm.

2 mg/k

14 21CONTROLS

7 14 2l doys

â€¢ft' t t

CHART1.â€”Carcinoma 1025 in mice after seven daily doses Similar destruction of the tumor resulted from half that dose,of 4 mg/kg of TSPA, showing complete destruction of tumors. Controls, no tumor regression.

suited in complete destruction of 7-day-old tumorsin about 70 per cent, whereas injections of 0.5 and0.25 mg/kg for 7 consecutive days caused muchless complete destruction of 7-day-old carcinomasand sarcomas. The average diameters of the Flexner-Jobling carcinoma and Jensen sarcoma, measured 7 days after implantation, were 8.5 X 5.5 X4 mm. and 20 X 9 X 7 mm., respectively.

Intraperitoneal injections of TSPA at a dose of2 mg/kg/day were begun when the tumors were 1day or 7 days old, and injections were continuedfor 7 days. The experiments showed that TSPAcaused complete inhibition of 1-day-old sarcomasin all cases, while all lymphosarcomas continued togrow rapidly. Under similar treatment, more than80 per cent of 7-day-old Jensen sarcomas regressed



46 Cancer Research

completely, while nearly all of the 7-day-old Murphy-Sturm lymphosarcomas in the same host grewnormally.

The above experiments were repeated withCarcinoma 1025 and Sarcoma MA 387, growingsimultaneously in the same mouse. Carcinoma1025 is sensitive, while Sarcoma MA 387 is resistant to TSPA. Repeated intraperitoneal injectionsof 4 mg/kg of TSPA caused complete destructionof 1- or 7-day-old carcinomas, but the compoundhad no effect on the sarcomas, all of which were

growing rapidly. In both experiments, then, theresponses of these tumors to the chemical agentwere the same whether they were growing alone indifferent animals or side by side in the same animal. These findings emphasize the specificity ofreaction of these tumors to this chemical agent.

DISCUSSIONIt is well known that the response of various

tumors to a given agent may be strikingly different(5, 12, 15, 17-19). The present results show that

EFFECT OF TSPA ON JENSEN SARCOMA IN RATS

2mg/k

7 14 21

l 0

4 f â€”

10 f â€” â€”

i em.

O.Smg/k

7 14 21CONTROLS

14

CHABT2.â€”Jensen sarcoma in rats after seven daily doses Similar destruction of the tumors resulted from i that dose,of 2 mg/kg of TSPA, showing complete destruction of tumors. Controls, rapid growth and no tumor regression.



TABLE 7APPROXIMATEPER CENTOFT-DAY-OLDMOUSEANDRATTUMORSCURED

BYVARIOUSPHOSPHORAMIDES*

Dose(mg/kg/day) Tumor

1-2.5 Flexner-Jobling carcinoma1-2.5 Walker carcinosarcoma 2561-2.5 Sarcoma R 39

1 Jensen sarcoma6 Carcinoma 10256 Ridgway osteogenic sarcoma6 Gardner lymphosarcoma1 Flexner-Jobling carcinoma1 Walker carcinosarcoma 2561 Sarcoma R 391 Jensen sarcoma4 Carcinoma 10254 Ridgway osteogenic sarcoma4 Gardner lymphosarcoma2 Flexner-Jobling carcinoma2 Walker carcinosarcoma 256

1-2 Jensen sarcoma* The approximate percentageof spontaneous tumor regressionsfor controls for the experiments in this table are: 2 per cent for Carcinoma

10^5,5 per cent for Ridgwayosteogenicsarcoma, 5 per cent for Gardner lymphosarcoma, 15per cent for Flexner-Joblingcarcinoma,2 per centfor Walker carcinosarcoma256,5 per cent for Sarcoma R 30, and 5 per cent for Jensen sarcoma.

TABLE 8EFFECTOFDIETHYLENEPHOSPHORAMIDEONWELLESTABLISHED7-DAY-OLDRATTUMORS*

Compound administeredDiethylene ph'osphoramide

Triethylene phosphoramide

Triethylene thiophosphoramide

No.treated7050403050804070008060607070SO60130No.cured5602624412532SI0284550554940SO112PerCentcures800658088380780937583787080608Â«

DOSENo. Av. WT. CHANGE

DEATHS TBEATED/CONTROLS RESULTS OF TREATMENT

1st week

Â±

Â±

2d weekTUMORSFlexner-Jobling carcinoma




Sarcoma R 39

Sarcoma R 39

Jensen sarcoma

Murphy-Sturm lymphosarcoma 1.0+22

* Ten animals in each group. The results presented are the average of combined groups.

TABLE 9EFFECTOFTRIETHYLENEPHOSPHORAMIDEONWELLESTABLISHED?-DAY-OLDMOUSEANDRATTUMORS*

(mg/kg/day)2.51.02.51.02.51.01.01.01stweek0/400/300/300/200/202/202/300/20(gm.)+4+

24+14+23+7+21+

15+35+7+25+10+24+7+28+

17

TUMORREGRESSION75 per cent at 2d week; 90 per

cent at 3d week

67 per cent at 4th week



60 per cent at 2d week; 100 percent at 3d week



No. Av. WT. CHANGE

DOSE DEATHS TREATED/CONTROLS RESULTS OP TREATMENT

2d week

+ + +

+ + +

+ + +

+ + +

+ + +

+

+ + +

+ + +

TDMOBCarcinoma1025Ridgway

osteogenic sarcomaGardnerlymphosarcomaGardner

lymphosarcomaFlexner-Jobling

carcinomaWalker

carcinosarcoma256Sarcoma

R39Jensen

sarcomaMurphy-Sturm

lymphosarcoma*

Ten animals in each group.(mg/kg/day)6.06.06.04.01.01.01.01.01.01st

week0/502/302/400/101/700/600/302/600/20(gm.)1stweÂ«-3+++1.5-3.5

+++1.5+0.5

+ +4+3+1

++-f+5+7

+++27+8

++27+17

+++30+

11+++24+8+

16The.esults presented are! the average of combined groups.

Tcuoa REGRESSIONS30 per cent at 2d week; 85 per

cent at 3d week


eS per cent at 1st week; 85 percent at 2d week

40 per cent at 1st week and 2dweek







48 Cancer Research

this holds true for diethylene phosphoramide, tri-ethylene phosphoramide, and triethylene thio-phosphoramide. These compounds produced complete and permanent regression of two mouse tumors, Ridgway osteogenic sarcoma and Gardnerlymphosarcoma (6C3HED), which hitherto havenot been amenable to chemical cure. Previously, ithas been found in our laboratories that Sarcoma

180 has been cured by 6-mercaptopurine and thatCarcinoma 1025 has been cured by 3-bis(]3-chloro-ethyl)aminomethyl-4-methoxymethyl-5-hydroxy-6-methyl pyridine and by triethylene melamine.These results encourage the hope that more chemicals may be found which have a destructive actionspecific for different tumors.

The differences in the effectiveness of DEPA,

TABLE10EFFECTOFTRIETHYLENETHIOPHOSPHORAMIDEONWELLESTABLISHED7-DAY-OLDMOUSEANDRATTUMORS*

TUMORSarcoma 180

Bashford carcinoma

Carcinoma 1025

Carcinoma 1025

Carcinoma 1025

Ridgway osteogenic sarcoma 4.0









Walker carcinosarcoma 256 2.0



Jensen sarcoma

Jensen sarcoma

Jensen sarcoma

Jensen sarcoma

Murphy-Sturm lymphosar- 2.0coma

Murphy-Sturm lymphosar- 1.0

DOSE(mgAg/day)4.04.04.02.01.0i

4.0i

2.0i

1.04.02.01.02.01.00.52.01.00.52.01.00.50.252.01.0No.DEA

TBS1st

week0/200/201/600/100/101/700/200/201/700/200/10*0/500/200/202/500/200/2015/902/400/200/200/300/10Av.

WT.CHANGETREATED/CONTROLS

RESULTS OFTREATMENT(gm.)1stweek-1.5

-+1.5-0.5

-+1-2.5

+++1.5-1.5

+++10

+++1-2.5

+++

1.50++1.5+1Â±+1â€”

1 +++4+2

Â±+4+5+7-2

+++26+13

+++18+22

++270

++35+8

++28+23

++31-1

+++200

+++26+7

+++26+23

++31+4+22+8+28Â«d

weekâ€”â€”+

+++

+++

+++++

+++â€”+

+++

++

++

+++

+++

+++

+++

+++

+â€”â€”Phe

results presented are the average of combined groups.

TuMOB REGRESSION




















SuGiURA AND STOCKâ€”Phospkoramides in a Tumor Spectrum 49

TEPA, and TSPA against the various experimental tumors merit discussion. Thus, both TEPA andTSPA are more effective against all four of themouse mammary carcinomas tested than againsttwo of the three sarcomas. Similarly, these compounds showed a greater inhibitory effect on thedevelopment of Ehrlich ascites carcinoma andKrebs 2 ascites carcinoma (both mammary cancerorigin) than on Sarcoma 180 ascites tumor. Otherepithelial tumors which responded well to treatment with TEPA and TSPA were Carcinoma1025, Grand epidermoid carcinoma, Lewis bladdercarcinoma, and Lewis lung carcinoma.

It is interesting to note that TEPA and TSPAhad a destructive effect on the well establishedRidgway osteogenic sarcoma but only a slightinhibitory effect on the Wagner osteogenic sarcoma. Both tumors arose spontaneously in AKRmice (17). Transplants of these tumors grow rapidly in AKR mice and kill animals in 3-4 weeks.Morphologically these tumors are similar. However, the alkaline phosphatase content of Ridgwayosteogenic sarcoma is less than that of Wagner osteogenic sarcomaâ€”about 20 units of alkalineglycerophosphatase/gm against about 50 units.

There is a striking difference in the response ofdifferent mouse lymphosarcomas toward the action of TEPA and TSPA. Although these compounds had a destructive effect on 7-day-oldGardner lymphosarcoma (about 60 per cent), theyhad relatively no effect on the growth of the Patterson and Mecca lymphosarcomas. Therefore,we attempted to discover any difference in biological behavior or in histological picture among themwhich might be responsible for the different sensitivity to antitumor action of TEPA and TSPA.Gardner lymphosarcoma arose in a C3H mouse(19), while the Patterson and Mecca lymphosarcomas arose in AKR mice (17). Histological examinations of these tumors revealed that Gardnerlymphosarcoma is composed almost exclusively oflarge immature lymphocytes, while the Pattersonand Mecca lymphosarcomas are composed almostexclusively of large lymphocytes. The Gardnerlymphosarcoma does not metastasize into theliver, spleen, or mesentery, although it metasta-sizes into the lymph nodes. On the other hand, thePatterson and Mecca lymphosarcomas metastasize into lymph nodes, spleen, liver, kidney, andmesentery, indicating that these lymphosarcomasare more malignant than the Gardner lymphosarcoma. The radiosensitivity of Gardner lymphosarcoma and Mecca lymphosarcoma in vivo appeared to be the same. It was found that a highpercentage of 1-day-old tumors irradiated with3,000 r failed to grow, whereas tumors irradiated

at a dose of 2,000 r grew in almost all cases. Thus,it is possible that the natural histories of the tumors contributed to the differences in their response to these agents.

In general, the rat tumors were more susceptible to the phosphoramides than the mouse tumors. This is similar to our findings with aminop-terin (16), A-methopterin (unpublished data),HN2 (17), 3-bis(/3-chloroethyl)aminomethyl-4-methoxymethyl-5-hydroxy-6-methylpyridine(17),triethylene melamine (19), and 1,9-dimethane-sulfonyloxynonane (12). Complete regression wasobtained in 30-100 per cent of well established 7-day-old tumors, namely, Flexner-Jobling carcinoma, Walker carcinosarcoma 256, Sarcoma R 39,and Jensen sarcoma, by administration of theabove-mentioned nine compounds. The Murphy-Sturm rat lymphosarcoma appeared insensitive tothe phosphoramides, as it did in all comparablestudies thus far (12,14,18,19). Particularly interesting is the fact that triethylene thiophosphor-amide resulted in 48 per cent cures of Walker carcinosarcoma 256, which was completely resistantto TEPA and DEPA, related chemicals. Replacement of the oxygen in the TEPA molecule by sulfur resulted in an increase in antitumor activity.

SUMMARY1. The effects of diethylene phosphoramide

(DEPA), triethylene phosphoramide (TEPA), andtriethylene thiophosphoramide (TSPA) have beentested against a spectrum of eighteen tumors of themouse, five tumors of the rat, and three ascites tumors of the mouse.

2. Daily maximum tolerated doses of 6 mg/kgof TEPA in mice had a destructive effect on Carcinoma 1025, Ridgway osteogenic sarcoma, andAndervont hepatoma; a marked inhibitory effecton Miyono adenocarcinoma and Gardner lymphosarcoma; a moderate inhibitory effect on Sarcoma180, Adenocarcinoma E 0771, Ehrlich carcinoma,Grand epidermoid carcinoma, Wagner osteogenicsarcoma, Lewis bladder carcinoma, and Lewis lungcarcinoma; a slight inhibitory effect on SarcomaMA 387, Bashford carcinoma 63, and Harding-Passey melanoma, but no effect on Sarcoma T 241.

3. Daily maximum tolerated doses of 4 mg/kgof TSPA in mice had a destructive effect on Carcinoma 1025, Ridgway osteogenic sarcoma, Gardner lymphosarcoma, and Andervont hepatoma; amarked inhibitory effect on Adenocarcinoma E0771 and Bashford carcinoma 63; a moderate inhibitory effect on Sarcoma 180, Miyono adenocarcinoma, Ehrlich carcinoma, Grand epidermoidcarcinoma, Wagner osteogenic sarcoma, Harding-Passey melanoma, Lewis bladder carcinoma, and



Cancer Research

Lewis lung carcinoma, and a slight inhibitory effect on Sarcoma T 241, Sarcoma MA 387, Patterson lymphosarcoma, and Mecca lymphosarcoma.

4. The effects of TEPA and TSPA on theseeighteen mouse tumors were generally similar, andthese compounds were far more effective thanDEPA.

5. Both TEPA and TSPA had a marked inhibitory effect upon the development of Ehrlichascites carcinoma and Krebs 2 ascites carcinoma,but only a slight inhibitory effect on Sarcoma 180ascites tumor. DEPA had relatively no effect onthese three ascites tumors.

6. The growth of spontaneous mammary ade-nocarcinomas in Swiss albino mice was stopped byrepeated injections of TSPA, but there was no significant increase in complete tumor regression overthe controls.

7. Daily doses of 1 mg/kg of DEPA, 1 mg/kg ofTEPA, and 2 mg/kg of TSPA in rats had a destructive effect on 1-day-old implants of Flexner-Jobling carcinoma, Walker carcinosarcoma 256,Sarcoma R 39, and Jensen sarcoma, but no effecton Murphy-Sturm lymphosarcoma.

8. Complete regression was obtained in largenumbers of certain well established 7-day-oldmouse and rat tumors by administration of sevendaily doses of DEPA, TEPA, and TSPA. For rats,daily doses of ]-2.5 mg/kg of DEPA gave 80 percent regressions for Flexner-Jobling carcinoma, 65per cent for Sarcoma R 39, and 80 per cent forJensen sarcoma. For mice, daily doses of 6 mg/kgof TEPA gave 82 per cent regressions for Carcinoma 1025, 83 per cent for Ridgway osteogenic sarcoma, and 80 per cent for Gardner lymphosarcoma. Similarly, for rats, daily doses of 1 mg/kg ofTEPA gave 73 per cent regressions for Flexner-Jobling carcinoma, 93 per cent for Sarcoma R 39,and 75 per cent for Jensen sarcoma. Similarly, for

mice, daily doses of 4 mg/kg of TSPA gave 83 percent regressions for Carcinoma 1025, 78 per centfor Ridgway osteogenic sarcoma, and 70 per centfor Gardner lymphosarcoma. For rats, daily dosesof 2 mg/kg gave 80 per cent regressions for Flexner-Jobling carcinoma, 60 per cent for Walker carcinosarcoma 256, and 86 per cent for Jensensarcoma.

REFERENCES1. BUCKLEY,S. M.; STOCK,C. C.; PARKER,R. P.; CHOSSLEY,

M. L.; KUH, E.; and SEEGER,D. R. Inhibition Studies ofSome Phosphoramides against Sarcoma 180. Proc. Soc.Exper. Biol. & Med., 78:299-305, 1951.

2. BURCHENAL,J. H.; JOHNSTON,S. F.; STOCK. C. C.;PARKER,R. P.; CROSSLEY,M. L.; KUH, E.; and SEEGEH,D. R. Effects of the N-Ethylene Substituted Phosphor-amides on Transplantable Mouse Leukemis. Cancer Research, 12:251-52, 1952.

3. CROSSLEY,M. L.; ALLISON,J. B.; PARKER,R. P.; KUH,E.; and SEEGER,D. R. Chemotherapy of Cancer in Rats.IV. The Treatment of Transplanted Cancers in Rats with\-Pentamethylene- and N-(3-oxapentamethylene)-N',X"-diethylene-phosphoramides. Cancer Research, 12:

256, 1952.4. FARBER,S.; APPLETON,R.; DOWNING,V.; HEALD, F.;

KING, J.; and TOCH,R. Clinical Studies on the Carcino-lytic Action of Triethylenephosphoramide. Cancer,6:135-41, 1953.

5. GELLHOHN,A.; ENGELMAN,M.; SHAPIRO,D.; GRAFF,S.;and GILLESPIE,H. The Effect of 5-Amino-7-hydroxy-Ãff-Ã¼-triazolo(d) Pyrimidine (Guanazolo) on a Varietyof Neoplasms in Experimental Animals. Cancer Research,10:170-77,1950.

6. PEHSONEUS,G.; HALLIDAY,S. L.; McKENziE, D.; andWILLIAMS,J. H. Effect of a Series of Ethylenimine Derivatives against Metastasizing Mammary Adenocarcinoma ofthe Rat. Proc. Soc. Exper. Biol. & Med., 81:614-16, 1952.

7. PHILIPS,F. S., and THIEKSCH,J. B. The Nitrogen Mustard-like Actions of 2,4,6-Tris(ethylenimino)-s-Triazine andOther Bis(ethylenimines). J. Pharmacol. & Exper. Therap.,100:398-407, 1950.

8. SHAY,H.; ZARAFONETIS,C.; SMITH,N.; WOLDOW,I.; andSUN, D. C. H. Treatment of Leukemia with TriethyleneThiophosphoramide (Thio-TEPA). Preliminary Results inExperimental and Clinical Leukemia. A.M.A. Arch. Inter.Med., 92:628-45, 1953.

FIG. 1.â€”Section of Grand mouse epidermoid carcinoma,showing early pearl formation. (H & E X200.)

FIG. 2.â€”Asection of the Lewis mouse bladder carcinoma,showing a compact sheet of cells having vesicular nuclei withirregularity in size and shape, and numerous mitoses. (X200.)

FIG. 3.â€”A section of the Lewis mouse lung carcinoma,showing a sheet of poorly differentiated epidermoid carcinoma.The tumor cells have large nuclei of irregular shape andvarying chromosia. (X200.)



,yÂ¿Â¿â€¢â€¢Â«v'Â¿â€¢..-:â€¢â€¢,â€¢;,/v-v..->â€¢â€ž.. â€¢.- - -.Â«. i' , ^ -.V:;. * ,V^:*;v*-,.VV-.^V^- 4-:V^



Fio. 4.â€”Sectionof untreated Jensen rat sarcoma, showingnumerous spindle cells with voluminous cytoplasm. (X200.)

FIG. 5.â€”Jensen sarcoma after treatment with TSPA(2 nig/kg for 7 days). Shows complete necrosis of the tumorcells (as seen in the lower right half of the picture). The surrounding tissue (upper left half) is made up of granulationtissue with a few lymphocytes, macrophages, and plasmacells. (X200.)



m>WM'3$3BÂ¿ 5?T-Ã•ES>-iÃ¯iifpÃ®iKsi'



STJGITIRAANDSTOCKâ€”Phosphoramidesin a Tumor Spectrum 51

9. SPARKS,S. J.; STEVENS,M. L.; LANDES,M. J.; HALLIDAT,S. L.; McKENziE, D.; and WILLIAMS,J. H. The Effect ofa Series of Ethylenimine Derivatives on Myeloid Chloro-leukemia in the Rat; Blood. J. Hematology, 8:655-60,1953.

10. SPARKS,S. J.; WALSH,M. E.; SKUASTIANELLI,L.; STEVENS, M.; LANDES,J.; and HALLIDAT,S. L. Chemotherapy of a Granulocytic Chloroleukemia in the Rat.Proc. Am. Assoc. Cancer Research, 1:46, 1954.

11. STOCK,C. C.; BUCKLEY,S. M.; CLARKE,D. A.; PARKER,R. P.; CHOSSLET,M. L.; Km, E.; and SEEGEH,D. R. Inhibitory Action of Some New Phosphoramides on Sarcoma180 in Mice. Cancer Research, 12:300,1952.

12. SUGIUHA.K. The Effect of 6-Thiopurine and of 1,9-Di-(methane sulfonoxy) nonane on the Growth of a Varietyof Mouse and Rat Tumors. Proc. Am. Assoc. Cancer Research, 1(1): 55, 1953.

13. . Effect of Various Compounds on the EhrlichAscites Carcinoma. Cancer Research, 13:431-41, 1953.

14. . The Effect of Thiotriethylene Phosphotamide onthe Growth of a Variety of Mouse and Rat Tumors.

Proc. Am. Assoc. Cancer Research, 1(2): 47-48, 1954.15. SUGIUHA,K.; HITCHINGS,G. H.; CAVALIERI,L. F.; and

STOCK,C. C. The Effect of 8-Azaguanine on the Growthof Carcinoma, Sarcoma, Osteogenic Sarcoma, Lympho-sarcoma, and Melanoma in Animals. Cancer Research,10:178-85, 1950.

16. SUGIURA,K.; MOORE,A. E.; and STOCK,C. C. The Effect

of Aminopterin on the Growth of Carcinoma, Sarcoma,and Melanoma in Animals. Cancer, 2:491-502, 1949.

17. SUGIURA,K., and STOCK,C. C. Studies in a Tumor Spectrum. I. Comparison of the Action of Methylbis(2-chlorethyl)amine and 3-bis(2-chlorethyl)aminomethyl-4-methoxymethyl-5-hydroxy-6-methylpyridine on theGrowth of a Variety of Mouse and Rat Tumors. Cancer,6:382-402, 1952.

18. . The Effect of Methylbis(0-chloroethyl)amineOxide, N,N',N"-Triethylene Phosphoramide andN,N-Diethyl-N', N"-Diethylene Phosphoramide on the

Growth of a Variety of Mouse and Rat Tumors. CancerResearch, 12:300-301, 1952.

19. . Studies in a Tumor Spectrum. II. The Effect of2,4,6-Triethylenimino-i-triazine on the Growth of a Variety of Mouse and Rat Tumors. Cancer, 6:979-91, 1952.

20. STKES, M. P.; KAHNOFSKT,D. A.; PHILIPS, F. S.; andBUHCHENAL,J. H. Clinical Studies on Triethylene-phos-phoramide and Diethylenephosphoramide, Compoundswith Nitrogen-like Activity. Cancer, 6:142-48, 1953.

21. WILLIAMS,J. H.; McKENziE, D.; HALLIDAT,S. L.; PEH-SONEÃœS,G. R.; STEVENS,M. L.; SPARKS,S. J.; SMITH,S. G.; TROT, W. P.; SCHURR,H. S.; GLEASON,H. R.;JAMES,E. R.; MOSER, L.; LTDICH,P.; LANDES,M. J.;EVE, V. ; STICK,P. ; and VINCENT,N. The Effect of a Seriesof Ethylene Amines against Experimental Cancer. CancerResearch, 12:310, 1952.



1955;15:38-51. Cancer Res Kanematsu Sugiura, C. Chester Stock and Miyono M. Sugiura on the Growth of a Variety of Mouse and Rat TumorsStudies in a Tumor Spectrum: III. The Effect of Phosphoramides

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